basis of -synuclein degradation: emerging support for multiple pathways basis of -synuclein...
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BASIS OF BASIS OF -SYNUCLEIN -SYNUCLEIN DEGRADATION:DEGRADATION:
Emerging Support for Multiple PathwaysEmerging Support for Multiple Pathways
Jessica PriceJessica Price
Advanced Cell and Molecular BiologyAdvanced Cell and Molecular Biology
Lake Forest CollegeLake Forest College
Road MapRoad Map
Introduction to Parkinson’s Introduction to Parkinson’s DiseaseDisease
-Synuclein Biology -Synuclein Biology Protein DegradationProtein Degradation HypothesisHypothesis ResultsResults ConclusionsConclusions Discussion & Future ResearchDiscussion & Future Research AcknowledgmentsAcknowledgments
SymptomsSymptoms
Resting tremor Resting tremor Muscular rigidityMuscular rigidity Postural instabilityPostural instability Slowed movementSlowed movement
Also called bradykinesiaAlso called bradykinesia
http://www.michaeljfox.org/news/article.php?id=17&sec=2
PathologyPathology
Death of dopaminergic Death of dopaminergic neurons neurons
Cytoplasmic inclusions of Cytoplasmic inclusions of misfolded misfolded -synuclein -synuclein called Lewy bodies called Lewy bodies
http://www.med.harvard.edu/AANLIB/cases/case11/mr1/011.gif
-Synuclein Biology-Synuclein Biology
14 kDa protein Abundant at presynaptic
terminals Precise function unknown Mutations facilitate Lewy
body aggregation in vitro
http://medweb.bham.ac.uk/http/depts/clin_neuro/teaching/tutorials/parkinsons/lewy.jpg
Two forms of PDTwo forms of PD
Mutations in:EnvironmentalFactors
Misfolding
Aggregation(Lewy bodies)
Toxicity
Cell Death
?
?
Sporadic (95%) Genetic (Familial) (5%)
•-Synuclein•UCHL-1•Parkin•Park 3•Pink-1
What happens to misfolded What happens to misfolded proteins?proteins?
Extracellular and membrane proteins
Proteins from the cytoplasm, nucleus, and ER
http://www.nature.com/nrm/journal/v1/n2/slideshow/nrm1100_145a_F1.html
Colin Gordon, www.hgu.mrc.ac.uk/Research/Gordon.
Ubiquitin-Proteasome SystemUbiquitin-Proteasome System
Lysosome SystemLysosome System
Lysosome
Figure 15-35. Biology 6th Edition, Campbell and Reece
All pathways involve vesicle mediated transport!
Evidence for the Ub-proteasomeEvidence for the Ub-proteasome
-Synuclein is characterized as a -Synuclein is characterized as a cytoplasmic proteincytoplasmic protein
-Synuclein is degraded by the -Synuclein is degraded by the ub-proteasome pathway ub-proteasome pathway (Bennet (Bennet et al., 1999; Holtz and O’Malley, 2003)et al., 1999; Holtz and O’Malley, 2003)
Mutations associated with PD inhibit Mutations associated with PD inhibit elements of the ub-proteasome pathway, elements of the ub-proteasome pathway, Parkin, PARK 3, & ubiquitin C-terminal Parkin, PARK 3, & ubiquitin C-terminal hydrolase L1 hydrolase L1 (Ceichanover and Brundin 2003; McNaught et (Ceichanover and Brundin 2003; McNaught et
al., 2002)al., 2002)
However,However,
Pharmacological studies have indicated Pharmacological studies have indicated that proteasome inhibitors do not alter that proteasome inhibitors do not alter cellular levels of α-synuclein cellular levels of α-synuclein (Rideout and Stefanis, (Rideout and Stefanis, 2002; Biasini et al., 2004)2002; Biasini et al., 2004)
-Synuclein has been shown to be -Synuclein has been shown to be translocated to lysosomes for degradation translocated to lysosomes for degradation (Cuervo et al., 2004)(Cuervo et al., 2004)
Wild type Wild type -Synuclein localizes to the cell -Synuclein localizes to the cell membrane in yeastmembrane in yeast
The Lysosome:The Lysosome: An alternate pathway? An alternate pathway?
Willingham, et. al. 2003 identified 86 genes that increase -synuclein toxicity
32% were involved with vesicle mediated transport and lipid metabolism
I chose to investigate Vps28
The MVB PathwayThe MVB Pathway
What does Vps28 do?What does Vps28 do?
Component of the ESCRT-1 complexComponent of the ESCRT-1 complex ESCRT-1 recognizes Ub-cargo at the endosome ESCRT-1 recognizes Ub-cargo at the endosome
and initiates transport of these cargos into and initiates transport of these cargos into vesicles that form MVBsvesicles that form MVBs
HypothesisHypothesis
The proteins composing the multivesicular The proteins composing the multivesicular body (MVB) sorting pathway play a key body (MVB) sorting pathway play a key role in the transport of role in the transport of -synuclein to the -synuclein to the lysosome for degradation.lysosome for degradation.
Aim 1:Aim 1:
Verify Verify -synuclein expression in cells -synuclein expression in cells lacking vps28lacking vps28
How?How?
Western AnalysisWestern Analysis
PredictionsPredictions
Method: Western AnalysisMethod: Western Analysis
For all transformants a single band is expected at approximately 58 kDa, corresponding to the monomeric form of -synuclein tagged with GFP, when expression was induced by galactose.
58 kDa
36 kDa
1 2 3 4 5 6 7 8 9 10 11 12 13 14
pYES2,
Gal
GFP, Gal
WT, G
al
WT, G
lu
A53T, G
al
A30P/A
53T, G
al
A30P, G
al
+ - + - + - + - + - + - + -
+ vps28
- vps28
1 2 3 4 5 6 7 8 9 10 11 12 13 14
-Synuclein is expressed in v-Synuclein is expressed in vps28ps28 strainsstrains
Aim 2:Aim 2:
Assess the impact of the lack of vps28 on Assess the impact of the lack of vps28 on growth of growth of -synuclein expressing cells-synuclein expressing cells
How?How?
Growth curve analysisGrowth curve analysis&&
Dilution series spottingDilution series spotting
Method: Grown Curve AnalysisMethod: Grown Curve Analysis
Growth in all transformants lacking vps28 will be inhibited by the production of -synuclein, indicated by higher cell densities in transformants with vps28.
Glucose Galactose24 h 24 h
Method: Growth Curve AnalysisMethod: Growth Curve Analysis
Prediction:Prediction:
Method: Growth Curve AnalysisMethod: Growth Curve Analysis
Evaluate OD over a period of 24h
Growth Curve of Cells With Vps28Growth Curve of Cells With Vps28
0
0.5
1
1.5
2
2.5
3
0 3 6 12 18 24
Time (hours)
Cel
l D
ensi
ty(A
bso
rban
ce a
t 60
0nm
)
WT
A30P
A43T
A30P/A53T
GFP
Empty Plasmid
WT Non-Induced
0
0.5
1
1.5
2
2.5
3
0 3 6 12 18 24
Time (hours)
Ce
ll D
en
sit
y
Growth Curve of Cells Lacking Growth Curve of Cells Lacking Vps28Vps28
Method: Dilution Series SpottingMethod: Dilution Series Spotting
5X Less 5X Less 5X Less
Prediction:Prediction:
Cells lacking vps28 will show inhibited growth when compared to the parent strain, with the mutant -synuclein transformants showing the most toxicity.
Non-Inducing Inducing
5x dilutions 5x dilutions
+
-
+
-
+
-
Vps28
WT α-synuclein
pYES2 Plasmid
GFP
Spotting Assessment of ToxicitySpotting Assessment of Toxicity
Spotting Assessment Cont.Spotting Assessment Cont.
Vps28
+
-
+
-
+
-
A30P α-synuclein
A53T α-synuclein
A30P/A53T α-synuclein
Non-Inducing Inducing
5x dilutions 5x dilutions
Aim 3:Aim 3:
Analyze the localization of Analyze the localization of -synuclein -synuclein
How?How?
GFP Fluorescence MicroscopyGFP Fluorescence Microscopy
PredictionsPredictions
Method: GFP Fluorescence Method: GFP Fluorescence MicroscopyMicroscopy
a-Synuclein will exhibit more cytosolic accumulation a-Synuclein will exhibit more cytosolic accumulation and aggregation in cells lacking vps28, with mutant a-and aggregation in cells lacking vps28, with mutant a-Synucleins demonstrating greater levels of Synucleins demonstrating greater levels of accumulation and aggregation.accumulation and aggregation.
+ vps28
- vps28
Wild Type A30P A53T A30P/A53T
Vps28Vps28 alters a-synuclein localization alters a-synuclein localization and increases aggregationand increases aggregation
Aim 4:Aim 4:
Assess the affect of vps28 absence on the Assess the affect of vps28 absence on the persistence and stability of cells persistence and stability of cells expressing a-synucleinexpressing a-synuclein
How?How?
Loss of Induction AssayLoss of Induction Assay
Method: Loss of Induction AssayMethod: Loss of Induction Assay
Glucose Galactose Glucose24 h 24 h
Western Analysis
24 h
Prediction:Prediction:
+ Vps28
-Vps28
0 2 4 6 8 10 12 14 16 18
Hours after Gal Shut-Off
58 kDa
58 kDa
0 .5 1 2 4 6 9 12 18 24
+ Vps28
-Vps28
Hours After Galactose Shut-Off
58 kDa
58 kDa
Vps28 Vps28 does not appear to affect does not appear to affect --synuclein stability over time synuclein stability over time
HypothesisHypothesis
The proteins composing the multivesicular The proteins composing the multivesicular body (MVB) sorting pathway play a key body (MVB) sorting pathway play a key role in the transport of α-synuclein to the role in the transport of α-synuclein to the lysosome for degradation.lysosome for degradation.
ConclusionsConclusions
The absence of vps28 increases a-The absence of vps28 increases a-synuclein toxicitysynuclein toxicity
Vps28 Vps28 leads to a-synuclein leads to a-synuclein accumulation accumulation in vivoin vivo
Vps28 presence does not discernibly Vps28 presence does not discernibly alter a-synuclein clearancealter a-synuclein clearance
Vps28 Absence increases a-Vps28 Absence increases a-Synuclein ToxicitySynuclein Toxicity
Increase in wild type a-synuclein toxicity previously been Increase in wild type a-synuclein toxicity previously been demonstrated in demonstrated in vps28vps28 in vivoin vivo by Willingham, et. al., by Willingham, et. al., 2003 confirmed2003 confirmed
A30P, A53T, and A30P/A53T mutant a-synuclein toxicity A30P, A53T, and A30P/A53T mutant a-synuclein toxicity was also modestly increased in the absence of vps28was also modestly increased in the absence of vps28
Absence variation in toxicity between wild type and Absence variation in toxicity between wild type and mutant a-synucleins implies that the absence of vps28 is mutant a-synucleins implies that the absence of vps28 is responsible for toxicity exclusively and not mutations in responsible for toxicity exclusively and not mutations in a-synuclein itself. a-synuclein itself.
This explains the sporadic occurrence of PD in patients This explains the sporadic occurrence of PD in patients that do not have a-synuclein mutations, tying sporadic that do not have a-synuclein mutations, tying sporadic PD to the accumulation of a-synuclein due to PD to the accumulation of a-synuclein due to dysfunctions in the vacuolar/lysosomal degradation dysfunctions in the vacuolar/lysosomal degradation pathway. pathway.
Vps28 Vps28 leads to a-synuclein leads to a-synuclein accumulation accumulation in vivoin vivo
Absence pf vps28 significantly alters the Absence pf vps28 significantly alters the localization of all a-synuclein forms and localization of all a-synuclein forms and increases the amount of a-synuclein cytoplasmic increases the amount of a-synuclein cytoplasmic inclusion inclusion
Presence of cytoplasmic inclusions of all forms Presence of cytoplasmic inclusions of all forms of a-synuclein in of a-synuclein in vps28 vps28 cells implies that the cells implies that the absence of vps28 leads to the accumulation of absence of vps28 leads to the accumulation of a-synuclein within the cell, a key aspect of PD. a-synuclein within the cell, a key aspect of PD.
The affect of vps28 on a-synuclein behavior The affect of vps28 on a-synuclein behavior points to the importance of the MVB pathway points to the importance of the MVB pathway and the lysosome in a-synuclein degradation. and the lysosome in a-synuclein degradation.
Vps28 presence does not discernibly Vps28 presence does not discernibly alter a-synuclein clearancealter a-synuclein clearance
Wild type a-synuclein persisted in both parent Wild type a-synuclein persisted in both parent strain and strain and vps28vps28 cells cells a-synuclein may be present in SDS-soluble a-synuclein may be present in SDS-soluble
aggregates which broke down to monomersaggregates which broke down to monomers Lack of vps28 may not be enough to increase a-Lack of vps28 may not be enough to increase a-
synuclein stability by a discernable amountsynuclein stability by a discernable amount Impact on wild type a-synuclein stability may not Impact on wild type a-synuclein stability may not
be dramatic enough to capture in this assay be dramatic enough to capture in this assay
DiscussionDiscussion
The Ub-Proteasome SystemThe Ub-Proteasome System The Lysosome SystemThe Lysosome System A New ModelA New Model
The Ub-Proteasome System:The Ub-Proteasome System:The Established PathwayThe Established Pathway
Ub-proteasome pathway degrades misfolded a-Ub-proteasome pathway degrades misfolded a-synuclein synuclein (Bennet et al., 1999; Holtz and O’Malley, 2003)(Bennet et al., 1999; Holtz and O’Malley, 2003)
Dysfunction of this pathway linked to a-synuclein Dysfunction of this pathway linked to a-synuclein accumulation and aggregation accumulation and aggregation (Sharma, 2004)(Sharma, 2004)
However, the function of the ubiquitin-However, the function of the ubiquitin-proteasome in clearing a-synuclein from the cell proteasome in clearing a-synuclein from the cell has been brought into question, implicating an has been brought into question, implicating an alternate method of a-synuclein degradation alternate method of a-synuclein degradation (Rideout and Stefanis, 2002; Biasini et al., 2004)(Rideout and Stefanis, 2002; Biasini et al., 2004)
The Lysosome: The Lysosome: The Emerging PathwayThe Emerging Pathway
a-Synuclein has also been shown to be targeted a-Synuclein has also been shown to be targeted to and degraded by the vacuole/lysosome to and degraded by the vacuole/lysosome (Cuervo (Cuervo
et al., 2004; Lee et al., 2004).et al., 2004; Lee et al., 2004). We demonstratedWe demonstrated
Disruption of this pathway elevates the toxicity of all Disruption of this pathway elevates the toxicity of all forms of a-synuclein forms of a-synuclein
Disruption of this pathway increase a-synuclein Disruption of this pathway increase a-synuclein accumulation and aggregation within cellsaccumulation and aggregation within cells
This indicates that disruption transport to the This indicates that disruption transport to the vacuole/lysosome for degradation has similar affects vacuole/lysosome for degradation has similar affects as the disruption of the ubiquitin-proteasome as the disruption of the ubiquitin-proteasome degradation pathway degradation pathway (Snyder et al., 2003, McNaught, et. al., (Snyder et al., 2003, McNaught, et. al., 2003)2003)
Misfolding Poly-Ub
Mono-Ub
Vacuole/LysosomeDegradation
MVB Pathway
α-Synuclein Ubiquitin
Established Pathway
Emerging Pathway
Two pathways work in conjunction Two pathways work in conjunction to degrade a-synuclein to degrade a-synuclein
Extracellular and membrane proteins
Proteins from the cytoplasm, nucleus, and ER
http://www.nature.com/nrm/journal/v1/n2/slideshow/nrm1100_145a_F1.html
Future ExperimentsFuture Experiments
Quantify aggregationQuantify aggregation Investigate other Vps proteinsInvestigate other Vps proteins
DOA4DOA4 Vps27Vps27
Confirmation of the ubiquitination state of Confirmation of the ubiquitination state of a-synuclein in a-synuclein in vps28vps28
Acknowledgments Acknowledgments
Dr. Shubhik DebBurmanDr. Shubhik DebBurmanIsaac HolmesIsaac HolmesNijee SharmaNijee Sharma
Katrina BrandisKatrina BrandisSara HerreraSara HerreraRuja ShresthaRuja ShresthaLavinia SinteanLavinia Sintean
Tasneem SaylawalaTasneem SaylawalaArun George PaulArun George Paul
NIHNIHNSFNSF
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